Active Caspase-3 Research Articles

Role of layilin in regulating mitochondria-mediated apoptosis: a study on B cell lymphoma (BCL)-2 family proteins

BackgroundMalignant gliomas exhibit rapid tumor progression and resistance to treatment, leading to high lethality. One of the causes is the reduced progression of apoptosis in glioma cells. Layilin is a type 1 transmembrane protein with a C-type lectin motif in its extracellular domain. We previously reported that layilin is mainly localized to mitochondria or their close proximity and that layilin is essential for maintaining of the fragmented type of mitochondria. This study investigates the effects of layilin on mitochondria-mediated apoptosis, focusing on B cell lymphoma (BCL)-2 family proteins in a glioma cell line of A172 cells.ResultsWe compared the levels of pro-apoptotic BCL-2 family proteins of BAD, BAK, BAX, and BIM and anti-apoptotic BCL-2 family proteins of BCL-2 and BCL-XL between layilin- knockdown (KD) cells and control cells using western blot. The protein levels of BAD were significantly smaller in layilin-KD cells than in control cells, while those of BCL-2 were significantly larger. We then compared the mitochondrial membrane potential (ΔΨm) under p-trifluoromethoxyphenyl hydrazone (FCCP)-treated conditions using MT-1 staining. In layilin-KD cells, ΔΨm was significantly larger and FCCP-induced ΔΨm reduction was significantly lower than in control cells. Furthermore, we examined the levels of cell membrane-bound Annexin V and DNA-bound propidium idodide (PI) in layilin-KD cells with/without staurosporine (STS) treatment. Layilin-KD significantly decreased levels of cell membrane-bound Annexin V with/without STS treatment. On the other hand, PI levels were not changed by layilin-KD. We also investigated the amounts of the active caspase (CASP)-3, CASP-6, CASP-7, and poly (ADP-ribose) polymerase-1 (PARP1, cleaved form), as well as DNA fragmentation in layilin-KD cells under apoptotic conditions induced by STS, using western blot and the DNA ladder method, respectively. Under STS-treated conditions, the amounts of active CASP-3, CASP-7, and poly (ADP-ribose) PARP1 were significantly smaller in layilin-KD cells than in control cells. Accordingly, DNA fragmentation was significantly suppressed in layilin-KD cells compared to control cells under STS-treated conditions.ConclusionThis study demonstrates that layilin contributes to ΔΨm reduction to promote apoptosis by up-regulating BAD and down-regulating BCL-2 in glioma cells. Our data elucidates a new function of layilin: regulation of mitochondria-mediated apoptosis.

Treatment of a mutant KRAS lung cancer cell line with polyisoprenylated cysteinyl amide inhibitors activates the MAPK pathway, inhibits cell migration and induces apoptosis.

KRAS mutations are the most common oncogenic mutations in lung adenocarcinoma in Black Americans. Polyisoprenylated Cysteinyl amide Inhibitors (PCAIs) constitute a group of potential cancer therapy agents that we designed to specifically disrupt and suppress hyperactive G-protein signaling, such as that caused by mutated RAS proteins. Here we determine the effects of PCAIs on the viability, G-protein levels, downstream mediators, and apoptosis-related proteins on the KRAS-mutated, Black American-derived lung adenocarcinoma cell line, NCI-H23. Of the 17 PCAIs tested, compounds NSL-YHJ-2-27 and NSL-YHJ-2-46 showed the most potency with EC50 values of 2.7 and 3.3 μM, respectively. Western blotting was used to determine the effect of the PCAIs on the phosphorylation levels of MAPK pathway enzymes. After 48 h exposure to 5 μM of the PCAIs, NSL-YHJ-2-46, the MAPK proteins BRAF, MEK1/2, ERK1/2, and p90RSK were activated through phosphorylation by 90, 190, 150 and 120%, respectively. However, CRAF/RAF1 phosphorylation decreased by 40%, suggesting significant changes in the KRAS/MAPK signaling patterns. Furthermore, 5 μM of NSL-YHJ-2-27 depleted the singly polyisoprenylated monomeric G-proteins RAC 1/2/3 and CDC42 by 77 and 76%, respectively. The depletion of these key cytoskeletal proteins may account for the observed inhibition of cell migration and invasion, and spheroid invasion observed on exposure to NSL-YHJ-2-27 and NSL-YHJ-2-46. Treatment with 5 μM of NSL-YHJ-2-27 suppressed full-length inactive caspase 3 and 7 levels by 72 and 91%, respectively. An analysis of cells treated with the fluorescently labeled active caspase 3/7 irreversible inhibitor, CaspaTagTM Caspase-3/7 in situ reagent revealed a 124% increase in active caspase at 3 μM over controls. These findings clearly show the direct effects of the PCAIs on the RAS signaling pathway. Given the profound increases observed in RPS6KA1/p90RSK phosphorylation, future work will involve a determination whether the proapoptotic isoforms of RPS6KA1/p90RSK are phosphorylated due to the PCAIs treatments. These results support the potential use of the PCAIs as targeted therapies against cancers with KRAS mutations.

Novel Strategies for Synthesis and Assessment of Dextran Conjugated 9Z,11E Linoleic Acid on Cancer Cell Lines Viability via Wnt/Beta‐Catenin Signaling Pathway and Apoptosis

AbstractConjugated linoleic acid (CLA) is one of the positional and structural isomers of linoleic acid. Ninety percent of the CLA contained in natural foods is the cis‐9(9Z), trans‐11(11E) isomer. CLA also has anti‐carcinogenic, anti‐adipogenic, and anti‐inflammatory properties. Limited studies aim to elucidate the impact of 9Z,11E‐CLA on Wnt/beta‐catenin signaling.In this study, we have selected MDA‐MB‐231, T98G, and SAOS‐2 cell lines of various cancer origins and investigate the impact of dextran conjugated 9Z,11E CLA (Dex‐9Z,11E‐CLA) using cell proliferation assay and immunocytochemical methods. Cytotoxic effects of Dex conjugated 9Z,11E CLA are assessed with the MTT method. It analyzed Wnt/beta‐catenin signaling changes using target molecule beta‐catenin, Dickkopf‐1 (Dkk‐1) as a negative regulator, and active caspase3 (CC3) as an apoptotic marker.Dex‐9Z,11E‐CLA is shown to suppress cell viability in three different types of cancer cells, and the most effective concentration is 200 µm. 9Z,11E‐CLA and Dex‐9Z,11E‐CLA affect the Wnt/beta‐catenin signaling pathway negatively in all cell lines. Beta‐catenin expression is decreased, Dkk‐1 expression is increased, and CC3 positivity is increased significantly compared to the control group. In conclusion, the data suggest that Dex‐9Z,11E‐CLA suppresses cell viability and causes apoptosis in cells via the Wnt/beta‐catenin signaling pathway. Lastly, Dex‐9Z,11E‐CLA harbors novel anticancer potential and exhibits uncontrolled activation of Wnt/beta‐catenin signaling.

Intermittent Fasting Ameliorates Age-Induced Morphological Changes in Aged Albino Rat Kidney via Autophagy Activation and Reduction of Apoptosis and Inflammation

Abstract Aging is a biological process with gradual decrease of cell function. Kidneys are one of the organs with higher susceptibility to the development of age-dependent tissue damage. Intermittent fasting has several beneficial effects on age-related degenerative changes. The aim of this study was to investigate the possible beneficial effect of intermittent fasting in delaying age-related renal changes and the possible mechanisms of this effect. Thirty male albino rats were classified into three groups: control, adult rats aged 3 months; aged group, 15-month-old rats and maintained until the age of 18 months; and intermittent fasting-aged groups, 15-month-old rats maintained on intermittent fasting for 3 months. Kidneys were processed for histological and immunohistochemical study. Aging resulted in a significant reduction in renal function and significant several degenerative changes in renal corpuscles and tubules which showed abnormal histological structure with increased collagen deposition. Aging caused significant reduction in the expression of autophagic marker light chain 3 with increased expression of active caspase-3 and inducible nitric oxide synthase. Intermittent fasting significantly improved these age-related renal changes. Intermittent fasting effectively prevents age-related renal changes through the reduction of age-related oxidative stress, inflammation, apoptosis, and activation of autophagy.

Design, synthesis and mechanistic study of N-4-Piperazinyl Butyryl Thiazolidinedione derivatives of ciprofloxacin with Anticancer Activity via Topoisomerase I/II inhibition

A new group of thiazolidine-2,4-dione derivatives of ciprofloxacin having butyryl linker 3a-l was synthesized via an alkylation of thiazolidine-2,4-diones with butyryl ciprofloxacin with yield range 48–77% andfully characterized by various spectroscopic and analytical tools. Anti-cancer screening outcomes indicated that 3a and 3i possess antiproliferative activities against human melanoma LOX IMVI cancer cell line with IC50 values of 26.7 ± 1.50 and 25.4 ± 1.43 µM, respectively, using doxorubicin and cisplatin as positive controls with an IC50 of 7.03 ± 0.40 and 5.07 ± 0.29 µM, respectively. Additionally, compound 3j showed promising anticancer activity against human renal cancer A498 cell line with IC50 value of 33.9 ± 1.91 µM while doxorubicin and cisplatin showed IC50 values of 3.59 ± 0.20 and 7.92 ± 0.45, respectively. On the other hand, compound 3i did not show considerable anti-bacterial activity against S. aureus, E. coli and P. aeruginosa, and only moderate activity against K. pneumoniae with only a tenth of the activity of ciprofloxacin, confirming the cytotoxicity observed. Mechanistically, compound 3i inhibited both topoisomerase I and II with IC50 of 4.77 ± 0.26 and 15 ± 0.81 µM. Furthermore, it induced cell cycle arrest at S phase in melanoma LOX IMVI cells. Moreover, 3i provoked substantial levels of early, late apoptosis and necrosis in melanoma LOX IMVI cell line comparable to that induced by doxorubicin. Furthermore, compound 3i increased the expression level of active caspase-3 by 49 folds higher in LOX IMVI cell, increased protein expression level of Bax more than the control by 3 folds and inhibited PARP-1by 33% in LOX IMVI. All results were supported by theoretical docking studies on both tested enzymes confirming potential cytotoxicity for the synthesized hybrids.

Clausena harmandiana root extract ameliorates Aβ1-42 induced cognitive deficits, oxidative stress, and apoptosis in rats

BackgroundClausena harmandiana (CH), commonly known as song fa dong, was a medicinal plant traditionally used to treat illnesses and as a health tonic. CH root extract (CHRE) exhibited various bioactivities, including neuroprotective, antioxidant, antimicrobial, antifungal, anti-inflammatory, and anti-cancer effects. However, CHRE data on neuroprotective in AD-like animal models were still scarce.ObjectivesThis study aimed to investigate the effects of CHRE on Aβ1-42-induced cognitive deficits, free radical damage, and neuronal death in rats.MethodsForty-eight adult male Sprague-Dawley rats (250–300 g) were classified as sham control (SC), V+Aβ, Vit C+Aβ, CHRE125+Aβ, CHRE250+Aβ, and CHRE500+Aβ (n = 8 in each group). Animals were orally administered with 0.5% sodium carboxymethylcellulose, vitamin C (200 mg/kg BW), or CHRE (125, 250, and 500 mg/kg BW) and were untreated for 35 days. On day 21, all treated rats were injected with 1 µl of aggregated Aβ1-42 (1 µg/µl) into the lateral ventricles, bilaterally, whereas untreated rats were injected with sterilized normal saline (NS). The Morris water maze test estimated the rat’s learning and memory one week later. At the end of the treatment, all rats were sacrificed, and their brains were removed and divided into two hemispheres. On the left, morphological changes and neuronal density were observed in hippocampal CA1 and CA3 regions. While, on the right, changes in free radical damage markers (SOD, CAT, GPx, MDA, and Nrf2) and protein expression of active caspase-3 were evaluated in the hippocampus.ResultsPretreatment with CHRE at all doses could alleviate spatial learning and memory defects. CHRE also improved morphological changes and a decrease in neuronal density in CA1 and CA3 regions. Additionally, CHRE significantly increased the activities of antioxidant enzymes (SOD, CAT, GPx) and Nrf2 expression. This was coupled with significantly decreased MDA levels and active caspase-3 expression in the hippocampus of Aβ1-42-induced rats, which was similar to vitamin C exposure.ConclusionsOur findings suggested that CHRE ameliorated cognitive deficits and exhibited neuroprotective effects by reducing free radical damage and mitigating neuronal abnormality and neuronal death.

6371 Conditional Deletion Of Nr5a1 In Sox9-cre Mice Impairs Adrenal Development

Abstract Disclosure: Y. Ikeda: None. A. Tagami: None. K. Ishizuka: None. M. Maekawa: None. The adrenal cortex is a major tissue that produces steroidogenic hormones necessary for regulating body homeostasis. Nuclear receptor subfamily 5, group A, member 1 (Nr5a1, SF-1, or Ad4BP) plays a crucial role in the regulation of adrenal and gonadal development, function and maintenance. SRY-related HMG-box gene 9 (Sox9) is upregulated by interaction of SRY with NR5A1, and leads the supporting cell precursors in XY gonads to differentiate into Sertoli-lineage cells at the initiation of testis differentiation. We previously generated Sox9-cre-Nr5a1flox/flox conditional knockout (cKO) mice, and showed that the loss of Nr5a1 in the gonads of XY Sox9-cre-Nr5a1flox/flox cKO mice abrogated the testicular pathway at embryonic day (E) 12.5–E13.5 to result in postnatal male-to-female gonadal sex reversal (1). Here, we investigated adrenal development of the Sox9-cre-Nr5a1flox/flox cKO mice. In mammals, after separation from the common adrenogonadal primordium, the adrenal primordium, which is formed dorsomedial to the gonadal primordium, begins to develop around E11.5. Double-immunostaining for NR5A1 and SOX9 revealed the presence of cells expressing both NR5A1 and SOX9 in the adrenal primordium of both sexes at E11.5 but not at E12.5. At E12.5, the number of NR5A1+ adrenocortical cells in Sox9-cre-Nr5a1flox/flox cKO mice markedly decreased compared with Nr5a1+/+ mice, and apoptotic cells, which were immunolabelled for active caspase 3, were detected in the area of the adrenal primordium in Sox9-cre-Nr5a1flox/flox cKO mice but not in Nr5a1+/+ mice, indicating that NR5A1- and SOX9-immunoreactive adrenocortical cells in Sox9-cre-Nr5a1flox/flox cKO mice were extinguished by apoptosis due to the activation of the Sox9-cre transgene. At E13.5–postnatal day 20, Sox9-cre-Nr5a1flox/flox cKO adrenals were hypoplastic associated with reduced number of NR5A1-immunoreactive adrenocortical cells, and only a few medullary cells immunolabelled for tyrosine hydroxylase, a marker of chromaffin cell precursors, were infiltrated into the adrenal primordium. Our findings suggest that sufficient number of adrenal cortical progenitors at the earliest stage of adrenal development are required for the subsequent adrenal development. In 8-week-old, Sox9-cre-Nr5a1flox/flox cKO adrenals were smaller in size compared with Nr5a1+/+ adrenals, but showed relatively normal organization with the medulla surrounded by the cortex. However, unlike XY Nr5a1+/+ adrenals, cells immunolabelled for the X-zone marker 20αHSD were observed at the junction between the cortex and medulla in XY Sox9-cre-Nr5a1flox/flox cKO adrenals, probably due to the sex reversal of the XY gonads, since androgen induces regression of the X-zone. Reference: (1) Ikeda et al., Sci Rep. 2021 Feb 24;11(1):4486. Presentation: 6/2/2024

Lymphoma cell-driven IL-16 is expressed in activated B-cell-like diffuse large Bcell lymphomas and regulates the pro-tumor microenvironment.

The activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) displays a worse outcome than the germinal center B-cell-like subtype (GCB-DLBCL). Currently, targeting tumor microenvironment (TME) is the promising approach to cure DLBCL with profound molecular heterogeneity, however, the factors affecting the tumor-promoting TME of ABCDLBCL are elusive. Here, cytokine interleukin-16 (IL-16) is expressed in tumor cells of ABCDLBCL and secreted by the cleavage of active caspase-3. The serum IL-16 levels are not only a sensitive marker of treatment response but also positively correlated with unfavorable prognosis in DLBCL patients. While IL-16 shows few direct promotional effects on tumor cell growth in vitro, its bioactive form significantly promotes tumor progression in vivo. Mechanically, IL-16 increases the infiltration of macrophages by the chemotaxis of CD4+ monocytes in the TME enhancing angiogenesis, and the expression of cytokine IL-6 and IL-10, as well as decreasing T cell infiltration to accelerate tumor progression. This study demonstrates that IL-16 exerts a novel role in coordinating the bidirectional interactions between tumor progression and the TME. IMM0306, a fusion protein of CD20 mAb with the CD47 binding domain of SIRPα, reverses the tumorpromoting effects of IL-16,which provides new insight into treatment strategy in ABC-DLBCL.

BV2 Microglial Cell Activation/Polarization Is Influenced by Extracellular Vesicles Released from Mutated SOD1 NSC-34 Motoneuron-like Cells.

Microglia-mediated neuroinflammation is a key player in the pathogenesis of amyotrophic lateral sclerosis (ALS) as it can contribute to the progressive degeneration of motor neurons (MNs). Here, we investigated the role of mSOD1 NSC-34 MN-like cell-derived extracellular vesicles (EVs) in inducing the activation of BV2 microglial cells. NSC-34-released EVs were isolated by culture medium differential ultracentrifugation to obtain two fractions, one containing small EVs (diameter < 200 nm) and the other containing large EVs (diameter > 200 nm). BV2 cells were incubated with the two EV fractions for 12, 24, and 48 h to evaluate 1) the state of microglial inflammation through RT-PCR of IL-1β, IL-6, IL-4, and IL-10 and 2) the expression of proteins involved in inflammasome activation (IL-β and caspase 1), cell death (caspase 3), and glial cell recruitment (CXCR1), and presence of the TGFβ cytokine receptor (TGFβ-R2). The obtained results suggest a mSOD1 type-dependent polarization of BV2 cells towards an early neurotoxic phenotype and a late neuroprotective status, with an appearance of mixed M1 and M2 microglia subpopulations. A significant role in driving microglial cell activation is played by the TGFβ/CX3CR1 axis. Therefore, targeting the dysregulated microglial response and modulating neuroinflammation could hold promise as a therapeutic strategy for ALS.

LRG1-Targeted Nintedanib Delivery for Enhanced Renal Fibrosis Mitigation.

Renal fibrosis lacks effective nephroprotective drugs in clinical settings due to poor accumulation of therapeutic agents in damaged kidneys, underscoring the urgent need for advanced renal-targeted delivery systems. Herein, we exploited the significantly increased expression of the leucine-rich α-2 glycoprotein 1 (LRG1) protein during renal fibrosis to develop a novel drug delivery system. Our engineered nanocarrier, DENNM, preferentially targets fibrotic kidneys via the decorated ET peptide's high affinity for LRG1. Once internalized by damaged renal cells, DENNM releases its encapsulated nintedanib, triggered by the active caspase-3 protease, disrupting the nanomedicine's structural integrity. The released nintedanib effectively reduces the level of expression of the extracellular matrix and impedes the progression of renal fibrosis by inhibiting the transforming growth factor-β (TGF-β)-Smad2/3 pathway. Our comprehensive in vitro and in vivo studies validate DENNM's antifibrotic efficacy, emphasizing LRG1's potential in renal targeted drug delivery and introducing an innovative approach to nanomedicine for treating renal fibrosis.

Mechanisms of retinal photoreceptor loss in spontaneously hypertensive rats

Retinal neurodegenerative diseases, including hypertensive retinopathy, involve progressive damage to retinal neurons, leading to visual impairment. In this study, we investigated the pathological mechanisms underlying retinal neurodegeneration in spontaneously hypertensive rats (SHR), using Wistar Kyoto (WKY) rats as normotensive controls. We observed that SHR exhibited significantly higher blood pressure and decreased retinal thickness, indicating retinal neurodegeneration. Molecular tests including quantitative real-time polymerase chain reaction, immunoblot, and immunofluorescent staining showed elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α, apoptotic markers (Fas, FasL, caspase-8, active caspase-3, and cleaved poly (ADP-ribose) polymerase), and necroptotic markers (receptor-interacting protein kinase-1 and -3) in SHR retinas. Additionally, we found elevated transforming growth factor-β (TGF-β) levels in the retinal pigment epithelium (RPE) of SHR, with a decrease in lecithin retinol acyltransferase (LRAT), which regulates retinoid metabolism and photoreceptor health. In human RPE cells (ARPE-19), TGF-β administration suppressed mRNA and protein levels of LRAT; and vactosertib, a selective inhibitor of TGF-β receptor kinase type 1, reversed the effect of TGF-β. These findings suggest that hypertension-induced retinal neurodegeneration involves inflammation, apoptosis, necroptosis, and disrupted retinoid metabolism, providing potential therapeutic targets for hypertensive retinopathy.

Pyroptosis leads to loss of centrosomal integrity in macrophages

NLRP3 forms a multiprotein inflammasome complex to initiate the inflammatory response when macrophages sense infection or tissue damage, which leads to caspase-1 activation, maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and Gasdermin-D (GSDMD) mediated pyroptosis. NLRP3 inflammasome activity must be controlled as unregulated and chronic inflammation underlies inflammatory and autoimmune diseases. Several findings uncovered that NLRP3 inflammasome activity is under the regulation of centrosome localized proteins such as NEK7 and HDAC6, however, whether the centrosome composition or structure is altered during the inflammasome activation is not known. Our data show that levels of the centrosomal scaffold protein pericentrin (PCNT) are reduced upon NLRP3 inflammasome activation via different activators in human and murine macrophages. PCNT loss occurs in the presence of membrane stabilizer punicalagin, suggesting this is not a consequence of membrane rupture. We found that PCNT loss is dependent on NLRP3 and active caspases as MCC950 and pan caspase inhibitor ZVAD prevent its degradation. Moreover, caspase-1 and GSDMD are both required for this NLRP3-mediated PCNT loss because absence of caspase-1 or GSDMD triggers an alternative regulation of PCNT via its cleavage by caspase-3 in response to nigericin stimulation. PCNT degradation occurs in response to nigericin, but also other NLRP3 activators including lysomotropic agent L-Leucyl-L-Leucine methyl ester (LLOMe) and hypotonicity but not AIM2 activation. Our work reveals that the NLRP3 inflammasome activation alters centrosome composition highlighting the need to further understand the role of this organelle during inflammatory responses.

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters.

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

RNA-based logic for selective protein expression in senescent cells

Cellular senescence is a cellular state characterized by irreversible growth arrest, resistance to apoptosis and secretion of inflammatory molecules, which is causally linked to the pathogenesis of many age-related diseases. Besides, there is accumulating evidence that selective removal of senescent cells can benefit therapies for cancer and fibrosis by modulating the inflammatory microenvironment. While the field of so-called senolytics has spawned promising small molecules and peptides for the selective removal of senescent cells, there is still no effective means to detect senescent cells in vivo, a prerequisite for understanding the role of senescence in pathophysiology and to assess the effectiveness of treatments aimed at removing senescent cells. Here, we present a strategy based on an mRNA logic circuit, that yields mRNA-dependent protein expression only when a senescence-specific miRNA signature is present. Following a validation of radiation-induced senescence induction in primary human fibroblasts, we identify miRNAs up- and downregulated in association with cellular senescence using RT-qPCR. Incorporating binding sites to these miRNAs into the 3’ untranslated regions of the mRNA logic circuit, we demonstrate the senescence-specific expression of EGFP for detection of senescent cells and of a constitutively active caspase-3 for selective removal. Altogether, our results pave the way for a novel approach to execute an mRNA-based programme specifically in senescent cells aimed at their detection or selective removal.

Galectin-3 protects distal convoluted tubules in rhabdomyolysis-induced kidney injury

Advanced glycation endproducts (AGEs) contribute to cellular damage of various pathologies, including kidney diseases. Acute kidney injury (AKI) represents a syndrome seldom characterized by a single, distinct pathophysiological cause. Rhabdomyolysis-induced acute kidney injury (RIAKI) constitutes roughly 15% of AKI cases, yet its underlying pathophysiology remains poorly understood. Using a murine model of RIAKI induced by muscular glycerol injection, we observed elevated levels of AGEs and the AGE receptor galectin-3 (LGALS3) in the kidney. Immunofluorescence localized LGALS3 to distal nephron segments. According to transcriptomic profiling via next-generation sequencing, RIAKI led to profound changes in kidney metabolism, oxidative stress, and inflammation. Cellular stress was evident in both proximal and distal tubules, as shown by kidney injury markers KIM-1 and NGAL. However, only proximal tubules exhibited overt damage and apoptosis, as detected by routine morphology, active Caspase-3, and TUNEL assay, respectively. In vitro, distal convoluted tubule (DCT) cells challenged with AGEs underwent apoptosis, which was markedly enhanced by Lgals3 siRNA treatment. Thus, in RIAKI, the upregulation of LGALS3 may protect the distal nephron from AGE-mediated damage, while proximal tubules lacking LGALS3 stay at risk. Thus, stimulating LGALS3 in the proximal nephron, if achievable, may attenuate RIAKI.

IPSC-induced neurons with the V337M MAPT mutation are selectively vulnerable to caspase-mediated cleavage of tau and apoptotic cell death

BackgroundTau post-translational modifications (PTMs) result in the gradual build-up of abnormal tau and neuronal degeneration in tauopathies, encompassing variants of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Tau proteolytically cleaved by active caspases, including caspase-6, may be neurotoxic and prone to self-aggregation. Also, our recent findings show that caspase-6 truncated tau represents a frequent and understudied aspect of tau pathology in AD in addition to phospho-tau pathology. In AD and Pick's disease, a large percentage of caspase-6 associated cleaved-tau positive neurons lack phospho-tau, suggesting that many vulnerable neurons to tau pathology go undetected when using conventional phospho-tau antibodies and possibly will not respond to phospho-tau based therapies. Therefore, therapeutic strategies against caspase cleaved-tau pathology could be necessary to modulate the extent of tau abnormalities in AD and other tauopathies. MethodsTo understand the timing and progression of caspase activation, tau cleavage, and neuronal death, we created two mAbs targeting caspase-6 tau cleavage sites and probed postmortem brain tissue from an individual with FTLD due to the V337M MAPT mutation. We then assessed tau cleavage and apoptotic stress response in cortical neurons derived from induced pluripotent stem cells (iPSCs) carrying the FTD-related V337M MAPT mutation. Finally, we evaluated the neuroprotective effects of caspase inhibitors in these iPSC-derived neurons. ResultsFTLD V337M MAPT postmortem brain showed positivity for both cleaved tau mAbs and active caspase-6. Relative to isogenic wild-type MAPT controls, V337M MAPT neurons cultured for 3 months post-differentiation showed a time-dependent increase in pathogenic tau in the form of caspase-cleaved tau, phospho-tau, and higher levels of tau oligomers. Accumulation of toxic tau species in V337M MAPT neurons was correlated with increased vulnerability to pro-apoptotic stress. Notably, this mutation-associated cell death was pharmacologically rescued by the inhibition of effector caspases. ConclusionsOur results suggest an upstream, time-dependent accumulation of caspase-6 cleaved tau in V337M MAPT neurons promoting neurotoxicity. These processes can be reversed by caspase inhibition. These results underscore the potential of developing caspase-6 inhibitors as therapeutic agents for FTLD and other tauopathies. Additionally, they highlight the promise of using caspase-cleaved tau as biomarkers for these conditions.

Caspase-3 promotes oncogene-induced malignant transformation via EndoG-dependent Src-STAT3 phosphorylation

Accumulating evidence suggests that caspase-3 plays critical roles beyond apoptosis, serving pro-survival functions in malignant transformation and tumorigenesis. However, the mechanism of non-apoptotic action of caspase-3 in oncogenic transformation remains unclear. In the present study, we show that caspase-3 is consistently activated in malignant transformation induced by exogenous expression of oncogenic cocktail (c-Myc, p53DD, Oct-4, and H-Ras) in vitro as well as in the mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT) mouse model of breast cancer. Genetic ablation of caspase-3 significantly attenuated oncogene-induced transformation of mammalian cells and delayed breast cancer progression in MMTV-PyMT transgenic mice. Mechanistically, active caspase-3 triggers the translocation of endonuclease G (EndoG) from mitochondria, which migrates to the nucleus, thereby induces phosphorylation of Src-STAT3 signaling pathway to facilitate oncogenic transformation. Taken together, our data suggest that caspase-3 plays pivotal role in facilitating rather than suppressing oncogene-induced malignant transformation of mammalian cells.

Ellagic acid mitigates heat-induced testicular detriment in a mouse model

Heat stress has been shown to have a detrimental impact on testicular activity and spermatogenesis. Ellagic acid is a plant-derived organic compound that has a variety of biological functions. Thus, it is believed that ellagic acid may improve heat-stressed testicular dysfunction. There has been no research on the impact of ellagic acid on heat-stressed testicular dysfunction. The mice were divided into 4 groups. The first group was the normal control group (CN), and the second received heat stress (HS) by submerging the lower body for 15 min in a water bath with a thermostatically controlled temperature kept at 43°C (HS), and the third and fourth groups were subjected to heat-stress similar to group two and given two different dosages of ellagic acid (5 mg/kg (EH5) and 50 mg/kg (EH50) for 14 days. Ellagic acid at a dose of 50 mg/kg improved the level of circulating testosterone (increased 3βHSD) and decreases the oxidative stress. The testicular and epididymal architecture along with sperm parameters also showed improvement. Ellagic acid treatment significantly increases the germ cell proliferation (GCNA, BrdU staining) and Bcl2 expression and decreases active caspase 3 expression. Heat stress downregulated the expression of AR, ER-α and ER-β, and treatment with ellagic acid increased the expression of ER-α and ER-β markers in the 50 mg/kg treatment group. Thus, our finding suggests that ellagic acid ameliorates heat-induced testicular impairment through modulating testosterone synthesis, germ cell proliferation, and oxidative stress. These effects could be manifested by regulating androgen and estrogen receptors. However, the two doses showed differential effects of some parameters, which require further investigation.

Mode of cell death in the penile cavernous tissue of type 1 diabetes mellitus rats.

Diabetes mellitus commonly causes endothelial cell and smooth muscle cell death in penile cavernous tissue. The study sought to study the mode of cell death in the penile cavernous tissue in type 1 diabetic rats. A total of 36 Sprague Dawley rats 10 weeks of age were randomly divided into 2 groups: a normoglycemic group and type 1 diabetic group (intraperitoneal injection of Streptozotocin (STZ), 60mg/kg). We randomly selected 6 rats from each group for tests at the end of 11, 14, and 18weeks of age, respectively. All rats were able to eat and drink freely. The ratio of maximum intracavernous pressure to mean arterial pressure, concentration of serum testosterone, level of nitric oxide in the penile cavernosum, and expression of active caspase-1 (pyroptosis) and active caspase-3 (apoptosis) were determined. At the end of weeks 4 and 8 of type 1 diabetes, the proportions of endothelial cells and smooth muscle cells undergoing apoptosis and pyroptosis in penile cavernous tissue are different. The ratio of maximum intracavernous pressure to mean arterial pressure and nitric oxide levels were significantly lower in the 4- and 8-week diabetic groups than in the normoglycemic group (P < .01). Penile endothelial cell pyroptosis (5.67 ± 0.81%), smooth muscle cell apoptosis (23.72 ± 0.48%), total cell pyroptosis (9.67 ± 0.73%), and total apoptosis (10.52 ± 1.45%) were significantly greater in the 4-week diabetic group than in the normoglycemic group (P < .01). The proportion of endothelial cell pyroptosis (24.4 ± 3.69%), endothelial cell apoptosis (22.13 ± 2.43%), total cell pyroptosis (14.75 ± 0.93%), and total apoptosis (14.82 ± 1.08%) in the penile tissues of the 8-week diabetic group were significantly greater than those in the normoglycemic group (P < .01).The 8-week survival proportions of diabetic endothelial cells (38.86 ± 8.85%) and smooth muscle cells (44.46 ± 2.94%) was significantly lower than the 4-week survival proportions of endothelial cells (93.17 ± 8.07%) and smooth muscle cells (75.12 ± 4.76%) (P < .05). Inhibition of cell death by different methods at different stages may be the key to the treatment of type 1 diabetes-induced erectile dysfunction. The effect of type 1 diabetes on other types of cell death in penile cavernous tissue needs further study. The mode of death of endothelial cells in the cavernous tissue of the penis in the early stage in diabetic rats is dominated by pyroptosis, and the death of smooth muscle cells is dominated by apoptosis. Endothelial cell and smooth muscle cell death are not consistent at different stages of diabetes progression.

Protection against α-Amanitin-induced liver toxicity: Efficacy of pomegranate seed oil and black cumin oil

The consumption of mushrooms containing α-Amanitin (α-A) can lead to severe liver damage. In this study, toxicological experiments were conducted to confirm the protective effects of pomegranate seed oil (PSO) and black cumin oil (BCO) against α-A-induced hepatotoxicity. Rats exposed once to α-A (3 mg/kg bw, i.p.) or saline alone (0.1 ml, i.p.) were either left untreated or treated with PSO or BCO at a dose of 2 ml/kg bw/day by oral gavage on the same day, and the treatment was continued for 7 days. Serum aminotransferases (ALT and AST), alkaline phosphatase (ALP) and total protein levels were measured and the active caspase 3 (cl-caspase 3) was evaluated by western blotting in the liver. Serum ALT, AST and ALP levels tended to decrease in the α-A exposed group, but no statistically significant difference was found compared to the saline group (p > 0.05). PSO and BCO did not affect serum liver function tests in rats exposed to saline or α-A. α-A toxicity was demonstrated by a significant decrease in serum total protein level (p < 0.05), a significant increase in liver cl-caspase 3 expression (p < 0.05), and structural liver damage mainly characterized by mononuclear inflammation and steatosis. When α-A exposed rats were treated with BCO, the increase in cl-caspase 3 was not inhibited, on the contrary BCO increased cl-caspase 3 in healthy rats (p < 0.05). PSO significantly ameliorated α-A-induced cl-caspase 3 increase and inflammatory histopathology in the liver. Both PSO and BCO completely prevented α-A-induced protein degradation. The findings indicate that PSO and BCO may protect liver functions against α-A-induced hepatotoxicity, encouraging future comprehensive studies to test them at different doses and frequency.